Regulatory T cells (Tregs) are a subset of CD4+ helper T cells that express the transcription factor Foxp3 and can suppress immune responses through a variety of mechanisms, including cytokine production and down-modulation of DC function. Under most conditions, Tregs do not proliferate, but we have previously shown that bone marrow dendritic cells (DCs) are specialized antigen presenting cells for stimulating proliferation and expansion of Tregs. Importantly, Tregs expanded by DCs maintain Foxp3 expression and are still suppressive in vitro and in vivo. Specifically, we showed that DC-expanded Tregs can reverse diabetes in NOD mice. We then hypothesized that, just as conventional T cells are optimally stimulated by DCs and antigen, Tregs stimulated with DCs would upregulate transcripts important for their suppressive function. Therefore, we have compared transcriptional profiles from Tregs stimulated with DCs, with profiles from resting Tregs or Tregs stimulated with IL-2. Interestingly, natural inhibitors of the IL-1 pathway were highly upregulated in Tregs stimulated by DCs. We are now studying the role of inhibitors of IL-1 for both Treg function and induction of new Tregs. In addition, transcriptional profiles of DCs that had been cultured with Tregs were compared to DCs cultured alone. As reported by others, we also observed downregulation of transcripts involved in antigen processing and presentation in DCs that had been cultured with Tregs. We have now focused on a small set of transcripts that are upregulated in DCs after culture with Tregs, but not after culture with nave helper T cells (CD4+ CD25- cells). We are beginning to test the role of these transcripts for DC function. For example, we have found that IL-1 Receptor antagonist (IL-1Ra) message is highly upregulated in Tregs after DC stimulation. To test if IL-1Ra is important for Treg function, we used Tregs from either wildtype or IL1Ra knockout mice to treat autoimmune colitis. We found that the knockout Tregs were not able to block further development of colitis, as measured by weight loss, histology, and IL-1 levels in the colon. We are currently following up on this finding.